GPCRs are remarkably versatile signaling molecules that are responsible for the majority of transmembrane signal transduction in response to hormones and neurotransmitters. They share a common structural signature of seven membrane-spanning helices with an extracellular amino terminus and an intracellular carboxyl terminus (FIG. 1). Our understanding of GPCR structure has been based largely on the crystal structures of the inactive state of rhodopsin. Rhodopsin is better suited for structural studies than most other GPCRs because it is possible to obtain large quantities of functional protein from bovine retina. Rhodopsin is also a remarkably stable GPCR, retaining function under conditions that denature other GPCRs.
The β2AR is one of the most extensively characterized members of this large family of membrane proteins. The sites of interactions between agonists and the receptor have been characterized by mutagenesis studies, and biophysical methods have been used to study the conformational changes associated with agonist binding and activation. The β2AR is efficiently expressed in Sf9 cells and can be purified to homogeneity using antibody and ligand affinity chromatography. The β2AR is biochemically pure following chromatography using an antibody resin that binds to an amino terminal Flag epitope; however, more than half of the receptor is not functional. Affinity chromatography, an important early development in GPCR biochemistry, may be used to isolate functional β2AR protein. Purified β2AR bound to an antagonist remains stable and soluble at concentrations up to 50 mg/ml for up to a week at room temperature in the detergent dodecylmaltoside. However, the β2AR is unstable in detergents used to obtain crystals of bovine rhodopsin. Extensive sparse matrix screening (over 2000 conditions at 4° and 20°) failed to produce diffraction-quality crystals of wild type β2AR.
This disclosure provides the atomic coordinates of human wild type β2AR.